Packages¶

In [1]:
import os
import time
import umap
import numpy as np
import scanpy as sc
import pandas as pd
import squidpy as sq
import seaborn as sns
import matplotlib.pyplot as plt
from collections import Counter
from scipy.stats import gaussian_kde

Diffusion matrix¶

In [2]:
%reload_ext memory_profiler
In [3]:
%%memit
diffusion_adata=sc.read_h5ad('/mnt/thechenlab/Mehdi/recon_umaps_c58_H4/H4_2_rec_merge/H4_2_rec_merge_filtered_.05p_log1p.h5ad')
diffusion_filtered = diffusion_adata[diffusion_adata.obs['n_counts'] > 0, diffusion_adata.var['n_counts'] > 0]
counts = diffusion_filtered.X

peak memory: 1950.94 MiB, increment: 1240.10 MiB
In [4]:
counts
Out[4]:
<878211x207289 sparse matrix of type '<class 'numpy.float32'>'
	with 47419940 stored elements in Compressed Sparse Row format>

Reconstruction with UMAP¶

In [ ]:
%%memit
n_epo = 10000

time5 = time.time()
reducer = umap.UMAP(metric='cosine',
                    n_neighbors=45, 
                    min_dist=0.4, 
                    low_memory=False, 
                    n_components=2, 
                    # random_state=0, 
                    verbose=True, 
                    n_epochs=n_epo,
                    # output_dens = True,
                    # local_connectivity = 30,
                    learning_rate = 1)
embedding = reducer.fit_transform(counts)
time_loop = time.time()
print('umap: ', time_loop-time5)

Slide-seq data stats¶

For reviewer: UEI per bead¶

In [ ]:
diffusion_filtered = sc.read_h5ad('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Data_Figures/Slide_recon_P1/H4_2_rec_merge_filtered_.05p_log1p.h5ad')
In [40]:
adata = sc.read_h5ad('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Data_Figures/Slide_recon_P1/adata_GeneFull_raw_matrix.h5ad')
In [41]:
a_df = pd.merge(adata.obs[['x','y']].copy(), diffusion_filtered.obs, left_index=True, right_index=True)
In [94]:
## FigS4k
fig = plt.figure(figsize=(5, 5))
ax = plt.axes()
im = ax.scatter(x=a_df.x, y=-a_df.y, c=a_df.n_counts, alpha=1, s=0.6, edgecolor='none',cmap='viridis')
plt.axis('equal')
plt.xlabel('X ($\mu$m)', fontsize=8)
plt.ylabel('Y ($\mu$m)', fontsize=8)
plt.tick_params(axis='both', which='major', labelsize=6)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
cax = fig.add_axes([ax.get_position().x1-0.01,ax.get_position().y0,0.02,ax.get_position().height])
cbar = plt.colorbar(im, cax=cax, pad=0.05, shrink=0.6)
cbar.ax.tick_params(labelsize=6, which='major') 
cbar.set_label('UEI per bead', fontsize=8)
# plt.subplots_adjust(left=0.1, right=0.8, top=0.8, bottom=0.1)
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4k_H4_2_uei_per_bead_spatial.png'),dpi=300)
plt.show()
No description has been provided for this image
In [47]:
plt.figure(figsize=(3, 2))
sns.histplot(a_df.n_counts, bins=50, alpha=0.7, edgecolor='black')
plt.xlabel('UEI per bead', fontsize=10)
plt.ylabel('number of beads', fontsize=10)
# plt.grid(True, linestyle='--', alpha=0.7)

plt.tick_params(axis='both', which='major', labelsize=8)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
plt.tight_layout()
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4m_H4_2_uei_per_bead_hist.png'),dpi=300)
plt.show()
No description has been provided for this image

For reviewer: before segmentation¶

In [34]:
adata = sc.read_h5ad('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Data_Figures/Slide_recon_P1/adata_GeneFull_raw_matrix.h5ad')
In [35]:
adata.obs['n_genes'] = adata.obs['n_genes'].replace([np.inf, -np.inf], np.nan)

# Then, remove rows where 'n_genes' is NaN
adata = adata[~adata.obs['n_genes'].isna()]

# Then, remove rows where 'n_genes' is NaN
adata = adata[~adata.obs['x'].isna()]
In [36]:
x = adata.obs.x.values
y = adata.obs.y.values
xmin, xmax = min(x), max(x)
ymin, ymax = min(y), max(y)
xx, yy = np.mgrid[xmin:xmax:100j, ymin:ymax:100j]
positions = np.vstack([xx.ravel(), yy.ravel()])
values = np.vstack([x, y])
kernel = gaussian_kde(values, weights=adata.obs.n_genes.values)
f = np.reshape(kernel(positions).T, xx.shape)
In [95]:
## FigS4l
fig = plt.figure(figsize=(5, 5))
ax = plt.axes()
im = ax.scatter(x=adata.obs.x, y=-adata.obs.y, c=adata.obs.log10_counts, alpha=1, s=0.6, edgecolor='none',cmap='viridis')
plt.axis('equal')
plt.xlabel('X ($\mu$m)', fontsize=8)
plt.ylabel('Y ($\mu$m)', fontsize=8)
plt.tick_params(axis='both', which='major', labelsize=6)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
cax = fig.add_axes([ax.get_position().x1-0.01,ax.get_position().y0,0.02,ax.get_position().height])
cbar = plt.colorbar(im, cax=cax, pad=0.05, shrink=0.6)
cbar.ax.tick_params(labelsize=6, which='major') 
cbar.set_label('log10(UMI per bead)', fontsize=8)
# cset = ax.contour(xx, -yy, f, colors='k',levels=[6.0e-09])
# plt.tight_layout()
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4l_H4_2_umi_per_bead_spatial_whole.png'),dpi=300)
# plt.close()
plt.show()
No description has been provided for this image

FigS9_a¶

gene expression library were analyzed by Slide-seq pipeline and RCTD

In [2]:
adata = sc.read_h5ad('/mnt/thechenlab/Chenlei/spotmapping/fiducial/data/H4_2/H4_2_mask_RCTD_result.h5ad')
In [9]:
fig = plt.figure(figsize=(6, 6))
ax = plt.axes()
im = ax.scatter(x=adata.obs.x, y=adata.obs.y, c=adata.obs.log10_counts, alpha=1, s=1, edgecolor='none',cmap='viridis')
plt.axis('equal')
plt.xlabel('X (µm)', fontsize=10)
plt.ylabel('Y (µm)', fontsize=10)
plt.tick_params(axis='both', which='major', labelsize=8)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
cax = fig.add_axes([ax.get_position().x1+0.01,ax.get_position().y0,0.02,ax.get_position().height])
cbar = plt.colorbar(im, cax=cax, pad=0.05, shrink=0.6)
cbar.ax.tick_params(labelsize=5, which='major') 
cbar.set_label('log10(UMI per bead)', fontsize=10)
# plt.tight_layout()
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4a_H4_2_umi_per_bead_spatial.png'),dpi=300)
# plt.close()
plt.show()
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FigS9_b¶

In [109]:
plt.figure(figsize=(3, 2))
sns.histplot(adata.obs.log10_counts, bins=50, alpha=0.7, edgecolor='black')
plt.xlabel('log10(UMI per bead)', fontsize=7)
plt.ylabel('number of beads', fontsize=7)
# plt.grid(True, linestyle='--', alpha=0.7)

plt.tick_params(axis='both', which='major', labelsize=5)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4b_H4_2_umi_per_bead_hist.svg'),dpi=300)
plt.show()
No description has been provided for this image

FigS9_c¶

In [2]:
palette = sns.color_palette(['#0d0305','#3c3444','#6e576e','#917d9b','#c5b7cb','#5f4f47','#851246','#d72048','#7d322f',
                             '#9d4c2f','#c65e2d','#f96a2d','#ffa300','#e29138','#f7c233','#11442c','#287a33','#52b139',
                             '#8ae931','#203c62','#2a69b0','#00a1de','#6bdad5','#a52eb8','#f7406e','#fc83a2','#f9cf9d','#fba176','#f66f67',])
In [19]:
type_order = ['Dermatome','Early chondrocytes','Definitive_erythroid','Adipocytes','Eye_and_other',
              'B_cells','Megakaryocytes','Olfactory_sensory_neurons','Fibroblasts','Facial mesenchyme','T_cells',
              'Chondrocytes (Otor+)','Ependymal_cells','Endothelium', 'Neuroectoderm_and_glia',
             'Intermediate_neuronal_progenitors','CNS_neurons','Epithelial_cells','Lateral plate and intermediate mesoderm',
             'Mast_cells','Neural_crest_PNS_glia','Neural_crest_PNS_neurons','Oligodendrocytes',
             'Lung_and_airway','Pre-osteoblasts (Sp7+)','Sclerotome','White_blood_cells','Muscle_cells','Chondrocytes (Atp1a2+)']
In [28]:
## FigS4c
fig = plt.figure(figsize=(4, 4))
sns.barplot(pd.DataFrame({'counts': Counter(adata.obs[adata.obs['spot_class'] == 'singlet']['first_type'])}).reset_index().rename(columns={'index': 'first_type'}),
           y='first_type',x='counts',hue='first_type', palette=palette, hue_order=type_order)
plt.xlabel('number of beads', fontsize=7)
plt.ylabel('cell type', fontsize=7)
plt.xscale('log')
plt.tick_params(axis='both', which='major', labelsize=5)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
plt.tight_layout()
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4c_H4_2_cell_type_bar.png'),dpi=300, bbox_inches='tight')
# plt.close()
plt.show()
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Slide-seq of P1 with reconstruction results¶

Fig2_f¶

In [182]:
plt.figure(figsize=(4, 4))
sns.scatterplot(adata.obs[adata.obs.spot_class != 'singlet'], x="x", y="y", color='#E3E3E3', s=0.1, edgecolor=None)
sns.scatterplot(adata.obs[adata.obs.spot_class == 'singlet'], x="x", y="y", hue="first_type", s=0.4, edgecolor=None, palette=palette, hue_order=type_order)

plt.axis('equal')
plt.legend(loc='upper left', frameon=False, bbox_to_anchor=(1, 1), fontsize=7)
plt.xlabel('X', fontsize=7)
plt.ylabel('Y', fontsize=7)
plt.tick_params(axis='both', which='major', labelsize=5)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4a_H4_2_cell_type_spatial.svg'),
#             dpi=300, bbox_inches='tight')
plt.show()
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Fig2_g¶

In [19]:
bead_umap = pd.DataFrame(adata.obsm['X_umap'], index=adata.obs.index, columns=['UMAP1','UMAP2'])
bead_umap = bead_umap.merge(adata.obs[['spot_class','first_type']], left_index=True, right_index=True)
In [126]:
plt.figure(figsize=(4, 4))

sns.scatterplot(bead_umap[bead_umap.spot_class != 'singlet'], x="UMAP1", y="UMAP2", color='#E3E3E3', s=0.1, edgecolor=None)
sns.scatterplot(bead_umap[bead_umap.spot_class == 'singlet'], x="UMAP1", y="UMAP2", hue="first_type", s=0.4, edgecolor=None, palette=palette, hue_order=type_order)

plt.axis('equal')
plt.legend(loc='upper left', frameon=False, bbox_to_anchor=(1, 1), fontsize=7)
plt.xlabel('UMAP1', fontsize=7)
plt.ylabel('UMAP2', fontsize=7)
plt.tick_params(axis='both', which='major', labelsize=5)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4b_H4_2_cell_type_umap.png'),
#             dpi=300, bbox_inches='tight')
plt.show()
No description has been provided for this image

Cell types¶

FigS10¶

In [155]:
fig, axs = plt.subplots(6, 5, figsize=(10, 10))

for i, ax in enumerate(axs.flat):
    if i < 29:
        ct = type_order[i]
        sns.scatterplot(adata.obs[(adata.obs.spot_class != 'singlet') | (adata.obs.first_type!=ct)]
                        , x="x", y="y", color='#F3F3F3', s=0.1, edgecolor=None, ax=ax)
        sns.scatterplot(adata.obs[(adata.obs.spot_class=='singlet') & (adata.obs.first_type==ct)]
                             , x="x", y="y", color=palette[i], s=0.2, edgecolor=None, ax=ax, legend=False)
        plt.axis('equal')
        ax.set_title(ct, fontsize=7, pad=10)
        ax.spines['top'].set_visible(False)
        ax.spines['bottom'].set_visible(False)
        ax.spines['left'].set_visible(False)
        ax.spines['right'].set_visible(False)
        ax.set_xticks([])
        ax.set_yticks([])
        ax.set_xlabel('')
        ax.set_ylabel('')
        
fig.delaxes(axs[5][4])
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4d_H4_2_cell_type_single.png'),dpi=300)
plt.tight_layout()
plt.show()
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Cell types and marker genes¶

In [ ]:
sc.tl.rank_genes_groups(adata, groupby='first_type', method='wilcoxon', n_jobs=20)
cluster_genes = pd.DataFrame(adata.uns['rank_genes_groups']['names'])

Fig2_h¶

In [127]:
fig, axs = plt.subplots(1, 4, figsize=(12, 3))

sns.scatterplot(adata.obs[(adata.obs.spot_class != 'singlet') | (adata.obs.first_type!='Adipocytes')]
                , x="x", y="y", color='#F3F3F3', s=0.1, edgecolor=None, ax=axs[0])
sns.scatterplot(adata.obs[(adata.obs.spot_class=='singlet') & (adata.obs.first_type=='Adipocytes')]
                     , x="x", y="y", color=palette[type_order.index('Adipocytes')], s=0.2, edgecolor=None, ax=axs[0], legend=False)
axs[0].axis('equal')
axs[0].set_title('Adipocytes', fontsize=7, pad=10)
axs[0].spines['top'].set_visible(False)
axs[0].spines['bottom'].set_visible(False)
axs[0].spines['left'].set_visible(False)
axs[0].spines['right'].set_visible(False)
axs[0].set_xticks([])
axs[0].set_yticks([])
axs[0].set_xlabel('')
axs[0].set_ylabel('')

neuronal_types = ['CNS_neurons','Neural_crest_PNS_neurons','Olfactory_sensory_neurons','Intermediate_neuronal_progenitors']
neuronal_palette = [palette[type_order.index(i)] for i in neuronal_types]
sns.scatterplot(adata.obs[(adata.obs.spot_class != 'singlet') | (~adata.obs['first_type'].isin(neuronal_types))]
                , x="x", y="y", color='#F3F3F3', s=0.1, edgecolor=None, ax=axs[1])
sns.scatterplot(adata.obs[(adata.obs.spot_class=='singlet') & (adata.obs.first_type.isin(neuronal_types))]
                     , x="x", y="y", palette=neuronal_palette,hue_order=neuronal_types, hue='first_type',
                s=0.2, edgecolor=None, ax=axs[1], legend=False)
axs[1].axis('equal')
axs[1].set_title('Neuronal cells', fontsize=7, pad=10)
axs[1].spines['top'].set_visible(False)
axs[1].spines['bottom'].set_visible(False)
axs[1].spines['left'].set_visible(False)
axs[1].spines['right'].set_visible(False)
axs[1].set_xticks([])
axs[1].set_yticks([])
axs[1].set_xlabel('')
axs[1].set_ylabel('')

sns.scatterplot(adata.obs[(adata.obs.spot_class != 'singlet') | (adata.obs.first_type!='Chondrocytes (Atp1a2+)')]
                , x="x", y="y", color='#F3F3F3', s=0.1, edgecolor=None, ax=axs[2])
sns.scatterplot(adata.obs[(adata.obs.spot_class=='singlet') & (adata.obs.first_type=='Chondrocytes (Atp1a2+)')]
                     , x="x", y="y", color=palette[type_order.index('Chondrocytes (Atp1a2+)')], s=0.2, edgecolor=None, ax=axs[2], legend=False)
axs[2].axis('equal')
axs[2].set_title('Chondrocytes (Atp1a2+)', fontsize=7, pad=10)
axs[2].spines['top'].set_visible(False)
axs[2].spines['bottom'].set_visible(False)
axs[2].spines['left'].set_visible(False)
axs[2].spines['right'].set_visible(False)
axs[2].set_xticks([])
axs[2].set_yticks([])
axs[2].set_xlabel('')
axs[2].set_ylabel('')

sns.scatterplot(adata.obs[(adata.obs.spot_class != 'singlet') | (~adata.obs['first_type'].isin(['Pre-osteoblasts (Sp7+)','Fibroblasts']))]
                , x="x", y="y", color='#F3F3F3', s=0.1, edgecolor=None, ax=axs[3])
sns.scatterplot(adata.obs[(adata.obs.spot_class=='singlet') & (adata.obs.first_type=='Fibroblasts')]
                     , x="x", y="y", color=palette[type_order.index('Fibroblasts')], s=0.2, edgecolor=None, ax=axs[3], legend=False)
sns.scatterplot(adata.obs[(adata.obs.spot_class=='singlet') & (adata.obs.first_type=='Pre-osteoblasts (Sp7+)')]
                     , x="x", y="y", color=palette[type_order.index('Pre-osteoblasts (Sp7+)')], s=0.2, edgecolor=None, ax=axs[3], legend=False)

axs[3].axis('equal')
axs[3].set_title('Fibroblasts, Osteoblasts', fontsize=7, pad=10)
axs[3].spines['top'].set_visible(False)
axs[3].spines['bottom'].set_visible(False)
axs[3].spines['left'].set_visible(False)
axs[3].spines['right'].set_visible(False)
axs[3].set_xticks([])
axs[3].set_yticks([])
axs[3].set_xlabel('')
axs[3].set_ylabel('')
        
plt.tight_layout()     
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4c_H4_2_cell_type_single4.png'),dpi=300)
plt.show()
No description has been provided for this image

Fig2_i¶

In [185]:
## Fig4d
marker_gene4 = ['Ucp1','Map1b','Ptgds','Col1a1']
# sc.settings.figdir = '/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4'
sc.pl.spatial(adata, color=marker_gene4, spot_size=20,
              cmap='Blues',size=1,frameon=False, colorbar_loc=None)#, save='Fig4d_maker_gene4.png')
plt.gcf().set_size_inches(12, 3)  # Set the figure size as width=10, height=8 inches
plt.show()

WARNING: saving figure to file /mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4/showFig4d_maker_gene4.png
No description has been provided for this image 
<Figure size 1200x300 with 0 Axes>

Neuronal cells¶

In [10]:
neuronal_types = ['CNS_neurons','Neural_crest_PNS_neurons','Olfactory_sensory_neurons','Intermediate_neuronal_progenitors']
adata_neuronal = adata[(adata.obs.spot_class == 'singlet') & (adata.obs.first_type.isin(neuronal_types))].copy()

FigS9_d¶

In [14]:
fig = plt.figure(figsize=(6, 6))
ax = plt.axes()
im = ax.scatter(x=adata_neuronal.obs.x, y=adata_neuronal.obs.y, c=adata_neuronal.obs.n_counts
                     , alpha=1, s=1, edgecolor='none',cmap='viridis')
plt.axis('equal')
plt.xlabel('X (µm)', fontsize=10)
plt.ylabel('Y (µm)', fontsize=10)
plt.tick_params(axis='both', which='major', labelsize=8)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
cax = fig.add_axes([ax.get_position().x1-0.02,ax.get_position().y0,0.02,ax.get_position().height])
cbar = plt.colorbar(im, cax=cax, pad=0.05, shrink=0.6)
cbar.ax.tick_params(labelsize=8, which='major') 
cbar.set_label('UMI per bead', fontsize=10)
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4e_H4_2_umi_per_bead_spatial_neuronal.png'),dpi=300)
# plt.close()
plt.show()
No description has been provided for this image

Fig2_j¶

In [41]:
fig, axs = plt.subplots(figsize=(5, 3))

clu_neuronal = ['2','3','5']
clu_label = ['Cortical neurons','Olfactory neurons','PNS neurons']
sns.scatterplot(adata_neuronal.obs[~adata_neuronal.obs.leiden.isin(clu_neuronal)], x="x", y="y", color='#D3D3D3', s=0.3, edgecolor=None)

for i in range(3):
    clu = clu_neuronal[i]
    sns.scatterplot(adata_neuronal.obs[(adata_neuronal.obs.leiden==clu)], x="x", y="y"
                    , color=adata_neuronal.uns['leiden_colors'][i], s=0.3, edgecolor=None, legend=False, label=clu_label[i])
plt.legend(loc='upper left', frameon=False, bbox_to_anchor=(1, 1), fontsize=7)
plt.axis('equal')
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)

plt.tight_layout()
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4e_H4_2_neuronal_spatial.svg'),dpi=300)
plt.show()
No description has been provided for this image

Fig2_k¶

In [187]:
fig, ax = plt.subplots(figsize=(3, 3))
neuronal_marker_gene = ['Mef2c', 'Ebf2', 'Sncg']
neuronal_palette = ['Blues', 'Oranges', 'Greens']

# Base layer scatter plot
sns.scatterplot(ax=ax, data=adata_neuronal.obs, x="x", y="y", color='#E3E3E3', s=0.4, edgecolor=None)

# Create a color bar for each gene
for i, ge in enumerate(neuronal_marker_gene):
    nonzero_bead = np.where(adata_neuronal.raw[:, ge].X.toarray().flatten() > 0)[0]
    # Extract the gene expression values
    ge_values = adata_neuronal.raw[:, ge].X.toarray().flatten()[nonzero_bead]
    # Create a scatter plot for each gene
    points = ax.scatter(adata_neuronal.obs.iloc[nonzero_bead]['x'], adata_neuronal.obs.iloc[nonzero_bead]['y'], 
                        c=ge_values, cmap=neuronal_palette[i], s=0.3, edgecolor='none', label=ge)
    
    # Create an axis for the color bar
    cax = fig.add_axes([0.95, 0.3+0.17*i, 0.02, 0.14])  # Adjust these values to position your color bar
    cbar = plt.colorbar(points, cax=cax, orientation='vertical')
    cbar.set_label(ge, fontsize=7)  # Set the label's fontsize
    cbar.ax.tick_params(labelsize=7)  # Set the tick's fontsize

ax.set_aspect('equal')
ax.spines['top'].set_visible(False)
ax.spines['right'].set_visible(False)
plt.tight_layout()
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4g_H4_2_neuronal_gene_spatial.svg'),dpi=300, bbox_inches='tight')
plt.show()

/tmp/ipykernel_7663/1225430776.py:27: UserWarning: This figure includes Axes that are not compatible with tight_layout, so results might be incorrect.
  plt.tight_layout()
No description has been provided for this image

FigS9_e¶

In [34]:
bead_umap_neuronal = pd.DataFrame(adata_neuronal.obsm['X_umap'], index=adata_neuronal.obs.index, columns=['UMAP1','UMAP2'])
bead_umap_neuronal = bead_umap_neuronal.merge(adata_neuronal.obs['leiden'], left_index=True, right_index=True)
In [40]:
plt.figure(figsize=(3.5, 3))

sns.scatterplot(bead_umap_neuronal[~bead_umap_neuronal.leiden.isin(clu_neuronal)], x="UMAP1", y="UMAP2", color='#D3D3D3', s=1, edgecolor=None)
for i in range(3):
    clu = clu_neuronal[i]
    sns.scatterplot(bead_umap_neuronal[bead_umap_neuronal.leiden==clu], x="UMAP1", y="UMAP2"
                    , color=adata_neuronal.uns['leiden_colors'][i], s=1, edgecolor=None, legend=False, label=clu_label[i])

plt.axis('equal')
plt.legend(loc='upper left', frameon=False, bbox_to_anchor=(1, 1), fontsize=7)
plt.xlabel('UMAP1', fontsize=7)
plt.ylabel('UMAP2', fontsize=7)
plt.tick_params(axis='both', which='major', labelsize=5)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4f_H4_2_neuronal_umap.svg'),dpi=300, bbox_inches='tight')
plt.show()
No description has been provided for this image

FigS9_f¶

In [196]:
fig, ax = plt.subplots(figsize=(3, 3))
neuronal_marker_gene = ['Mef2c', 'Ebf2', 'Sncg']
neuronal_palette = ['Blues', 'Oranges', 'Greens']

# Base layer scatter plot
sns.scatterplot(ax=ax, data=bead_umap_neuronal, x="UMAP1", y="UMAP2", color='#E3E3E3', s=0.4, edgecolor=None)

# Create a color bar for each gene
for i, ge in enumerate(neuronal_marker_gene):
    nonzero_bead = np.where(adata_neuronal.raw[:, ge].X.toarray().flatten() > 0)[0]
    # Extract the gene expression values
    ge_values = adata_neuronal.raw[:, ge].X.toarray().flatten()[nonzero_bead]
    # Create a scatter plot for each gene
    points = ax.scatter(bead_umap_neuronal.iloc[nonzero_bead]["UMAP1"], bead_umap_neuronal.iloc[nonzero_bead]["UMAP2"], 
                        c=ge_values, cmap=neuronal_palette[i], s=0.06, label=ge)
    
    # Create an axis for the color bar
    cax = fig.add_axes([0.95, 0.3+0.17*i, 0.02, 0.14])  # Adjust these values to position your color bar
    cbar = plt.colorbar(points, cax=cax, orientation='vertical')
    cbar.set_label(ge, fontsize=7)  # Set the label's fontsize
    cbar.ax.tick_params(labelsize=7)  # Set the tick's fontsize

ax.set_aspect('equal')
ax.spines['top'].set_visible(False)
ax.spines['right'].set_visible(False)
plt.tight_layout()
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4h_H4_2_neuronal_gene_umap.svg'),dpi=300, bbox_inches='tight')
plt.show()

/tmp/ipykernel_7663/931630624.py:27: UserWarning: This figure includes Axes that are not compatible with tight_layout, so results might be incorrect.
  plt.tight_layout()
No description has been provided for this image

Olfactory epithelium¶

In [20]:
palette = sns.color_palette(['#0d0305','#3c3444','#6e576e','#917d9b','#c5b7cb','#5f4f47','#851246','#d72048','#7d322f',
                             '#9d4c2f','#c65e2d','#f96a2d','#ffa300','#e29138','#f7c233','#11442c','#287a33','#52b139',
                             '#8ae931','#203c62','#2a69b0','#00a1de','#6bdad5','#a52eb8','#f7406e','#fc83a2','#f9cf9d','#fba176','#f66f67',])
In [21]:
Olfactory_types = ['Olfactory_sensory_neurons','Ependymal_cells','Epithelial_cells','Lung_and_airway']

adata_olfactory = adata[(adata.obs.spot_class == 'singlet') & (adata.obs.first_type.isin(Olfactory_types))].copy()
adata_olfactory = adata_olfactory[(adata_olfactory.obs.x > 2000) & (adata_olfactory.obs.x < 6000) & (adata_olfactory.obs.y > 2000) & (adata_olfactory.obs.y < 5000)]

olfactory_palette = [palette[7],palette[14],palette[17],palette[21]]

FigS11_a¶

In [28]:
marker_gene4 = ['Cbr2','Reg3g','Gap43','Cyp2g1']
sc.settings.figdir = '/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4'
sc.pl.spatial(adata_olfactory, color=marker_gene4, spot_size=20,
              cmap='Blues',size=1,frameon=False, show=False,ncols=4)
plt.gcf().set_size_inches(12, 2.5)  # Set the figure size as width=10, height=8 inches
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4e_H4_2_olfactory_4genes.png'),dpi=300)
plt.show()
No description has been provided for this image
In [14]:
adata_olfactory.var['means_olfactory'] = np.mean(adata_olfactory.X, axis=0)

/tmp/ipykernel_932772/3201788258.py:1: ImplicitModificationWarning: Trying to modify attribute `.var` of view, initializing view as actual.
  adata_olfactory.var['means_olfactory'] = np.mean(adata_olfactory.X, axis=0)
In [15]:
adata_olfactory.var['olfactory_enrich'] = adata_olfactory.var['means_olfactory'] / adata_olfactory.var['means']
In [53]:
adata_olfactory.var.sort_values(by='olfactory_enrich',ascending=False).head(30)
Out[53]:
n_cells n_counts log10_counts log10_cells mt n_cells_by_counts mean_counts pct_dropout_by_counts total_counts highly_variable means dispersions dispersions_norm mean std means_olfactory olfactory_enrich
Sec14l3 2714 4173.0 3.620448 3.464340 False 2714 0.008197 99.438946 3965.0 True 0.294692 5.009099 0.439364 6.439494e-12 0.287471 1.456197 4.941428
Ccdc153 1684 2279.0 3.357744 3.272538 False 1684 0.004304 99.651873 2082.0 True 0.200324 5.078300 0.557020 -2.092016e-11 0.229576 0.979683 4.890506
Fstl5 2212 4699.0 3.672005 3.536306 False 2212 0.007157 99.542722 3462.0 True 0.206661 5.147693 0.675003 -1.040028e-11 0.242731 0.867445 4.197435
Dynlrb2 1822 2371.0 3.374932 3.295787 False 1822 0.004577 99.623345 2214.0 True 0.213852 5.120139 0.628156 -1.459488e-11 0.237618 0.873337 4.083839
Cyp2f2 3681 5962.0 3.775392 3.620448 False 3681 0.011281 99.239041 5457.0 True 0.396482 5.106511 0.913117 2.993587e-11 0.338124 1.323024 3.336905
Lypd2 2566 3394.0 3.530712 3.464936 False 2566 0.006276 99.469541 3036.0 True 0.330134 5.274927 1.908146 -3.785615e-12 0.291782 0.983983 2.980554
Bpifb9a 2692 10526.0 4.022264 3.515079 False 2692 0.020515 99.443494 9924.0 True 0.638465 5.634588 4.542959 -1.133573e-11 0.360982 1.644382 2.575524
Aldh1a1 3601 5215.0 3.717254 3.617000 False 3601 0.009640 99.255580 4663.0 True 0.414126 5.071374 0.705525 7.312334e-12 0.343267 1.063272 2.567508
Krt19 3517 5149.0 3.711723 3.596597 False 3517 0.009722 99.272945 4703.0 True 0.400711 5.082903 0.773639 7.533710e-12 0.337738 1.012499 2.526758
Gng13 1494 1969.0 3.294246 3.245019 False 1494 0.003514 99.691151 1700.0 True 0.206695 5.240065 0.832055 -1.401129e-11 0.221342 0.486405 2.353247
Wfdc2 2392 2959.0 3.471145 3.418467 False 2392 0.005623 99.505511 2720.0 True 0.271303 5.079014 0.558234 -7.769679e-12 0.271986 0.624358 2.301330
Bpifb5 3610 15013.0 4.176467 3.676328 False 3610 0.028611 99.253719 13840.0 True 0.836769 5.680536 4.846414 2.359774e-11 0.424590 1.895954 2.265803
Ccdc181 2389 2883.0 3.459845 3.411283 False 2389 0.005567 99.506131 2693.0 True 0.251704 5.126059 0.638220 -1.448589e-11 0.265866 0.568353 2.258019
Krt18 4173 5315.0 3.725503 3.669131 False 4173 0.009939 99.137332 4808.0 True 0.394673 5.029976 0.460939 8.678897e-12 0.348511 0.875944 2.219420
Cyp2g1 1950 4161.0 3.619198 3.345374 False 1950 0.008042 99.596884 3890.0 True 0.296288 5.208218 0.777908 6.877138e-12 0.265270 0.654248 2.208152
Dnah9 2546 5342.0 3.727704 3.652053 False 2546 0.006987 99.473676 3380.0 True 0.352041 5.499484 3.234863 -3.894743e-12 0.289628 0.757793 2.152568
Med12l 2537 4244.0 3.627775 3.574610 False 2537 0.006233 99.475536 3015.0 True 0.213040 5.171462 0.715415 -6.075105e-12 0.254182 0.450883 2.116427
Cbr2 12844 25529.0 4.407034 4.161937 False 12844 0.049217 97.344811 23808.0 True 1.248648 5.286570 1.471365 -2.710992e-11 0.685302 2.410705 1.930652
Olfm5 2074 6081.0 3.783975 3.379124 False 2074 0.011899 99.571250 5756.0 True 0.440766 5.529126 3.409992 7.808372e-12 0.300485 0.816553 1.852575
Agr2 2124 2640.0 3.421604 3.357363 False 2124 0.005137 99.560914 2485.0 True 0.266190 5.259772 0.865561 -8.828838e-12 0.261958 0.486505 1.827659
Spint2 5200 6252.0 3.796019 3.761853 False 5200 0.011705 98.925025 5662.0 True 0.454734 5.028783 0.453888 5.254595e-11 0.380630 0.823590 1.811148
Cntn4 3474 7563.0 3.878694 3.769303 False 3474 0.010615 99.281834 5135.0 True 0.389527 5.280003 1.938133 -9.846770e-12 0.326410 0.650907 1.671018
Cdh1 3266 7509.0 3.875582 3.837273 False 3266 0.007934 99.324833 3838.0 True 0.290731 5.031961 0.478235 -1.013977e-11 0.296496 0.471788 1.622764
Cds1 2593 3084.0 3.489114 3.451786 False 2593 0.005875 99.463959 2842.0 True 0.245759 5.071997 0.546305 6.626148e-12 0.265341 0.389272 1.583961
Atf5 5219 6639.0 3.822103 3.762303 False 5219 0.012540 98.921097 6066.0 True 0.481112 5.089707 0.813835 -8.188369e-12 0.385363 0.741277 1.540760
Pifo 1540 2368.0 3.374382 3.356790 False 1540 0.003376 99.681642 1633.0 True 0.224766 5.416316 1.131719 -1.685941e-11 0.226313 0.338171 1.504549
Agbl4 2669 4841.0 3.684935 3.649432 False 2669 0.006278 99.448248 3037.0 True 0.323974 5.455776 2.976625 2.071100e-11 0.284320 0.479629 1.480455
Lhx2 3541 4527.0 3.655811 3.586925 False 3541 0.008678 99.267983 4198.0 True 0.315000 5.022155 0.461563 -1.406190e-11 0.309362 0.459973 1.460231
Bpifb9b 5102 36941.0 4.567509 3.856910 False 5102 0.071922 98.945284 34791.0 True 1.398609 6.411414 5.387797 5.238148e-13 0.535356 1.970066 1.408589
Dcxr 1725 2059.0 3.313656 3.291813 False 1725 0.003769 99.643398 1823.0 True 0.212073 5.138232 0.658918 3.071642e-12 0.232342 0.297270 1.401733

FigS11_b¶

In [29]:
## FigS4f
marker_gene4 = ['Sec14l3','Olfm5','Fstl5','Dcxr']
sc.settings.figdir = '/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4'
sc.pl.spatial(adata_olfactory, color=marker_gene4, spot_size=20,
              cmap='Blues',size=1,frameon=False, show=False,ncols=4)
plt.gcf().set_size_inches(12, 2.5)  # Set the figure size as width=10, height=8 inches
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4f_H4_2_olfactory_4moregenes.png'),dpi=300)
plt.show()
No description has been provided for this image

Epithelial cells¶

In [15]:
adata_epithelial = adata[(adata.obs.spot_class == 'singlet') & (adata.obs.first_type == 'Epithelial_cells')].copy()

FigS9_g¶

In [17]:
fig = plt.figure(figsize=(6, 6))
ax = plt.axes()
im = ax.scatter(x=adata_epithelial.obs.x, y=adata_epithelial.obs.y, c=adata_epithelial.obs.n_counts, alpha=1, s=1, edgecolor='none',cmap='viridis')
plt.axis('equal')
plt.xlabel('X (µm)', fontsize=10)
plt.ylabel('Y (µm)', fontsize=10)
plt.tick_params(axis='both', which='major', labelsize=8)
plt.gca().spines['top'].set_visible(False)
plt.gca().spines['right'].set_visible(False)
cax = fig.add_axes([ax.get_position().x1-0.02,ax.get_position().y0,0.02,ax.get_position().height])
cbar = plt.colorbar(im, cax=cax, pad=0.05, shrink=0.6)
cbar.ax.tick_params(labelsize=8, which='major') 
cbar.set_label('UMI per bead', fontsize=10)
plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4f_H4_2_umi_per_bead_spatial_epithelial.png'),dpi=300)
# plt.close()
plt.show()
No description has been provided for this image
In [ ]:
epi_de = pd.read_csv('/mnt/thechenlab/Chenlei/spotmapping/fiducial/data/H4_2/olfactory_de/de_summary/sig_Epithelial_cells.csv')
epi_de.columns = ['gene'] + list(epi_de.columns[1:])
In [18]:
epi_de_gene20 = epi_de.gene[:20].values
sq.gr.spatial_neighbors(adata_epithelial)
sq.gr.spatial_autocorr(
    adata_epithelial,
    mode="moran",
    genes=epi_de_gene20,
    n_perms=100,
    n_jobs=10,
    use_raw=True
)

moranI_gene20 = [adata_epithelial.uns["moranI"].loc[gene,'I'] for gene in epi_de_gene20]

Fig2_l¶

In [75]:
from matplotlib.lines import Line2D
fig, ax1 = plt.subplots(figsize=(4, 2))
sns.stripplot(data=epi_de.iloc[:20,], x="gene", y="Z_score", ax=ax1, c=sns.color_palette("tab10")[0], alpha=0.9)
ax1.set_ylabel('C-SIDE Z-score', fontsize=7)
ax1.set_xticklabels(ax1.get_xticklabels(), rotation=50)
ax1.set_xlabel('Epithelial Specific Gene', fontsize=7)
ax1.tick_params(axis='both', which='major', labelsize=5)
ax1.spines['top'].set_visible(False)
ax1.spines['right'].set_visible(False)

ax2 = plt.twinx()
sns.stripplot(x=epi_de.iloc[:20,].gene, y=moranI_gene20, ax=ax2, c=sns.color_palette("tab10")[1], alpha=0.7)
ax2.set_ylabel("Moran's I", fontsize=7)
ax2.tick_params(axis='both', which='major', labelsize=5)
ax2.spines['top'].set_visible(False)
ax2.spines['left'].set_visible(False)

# Create custom Line2D objects as legend keys to resemble dots
legend_dot1 = Line2D([0], [0], marker='o', color='w', label='C-SIDE Z-score',
                     markerfacecolor=sns.color_palette("tab10")[0], markersize=5, alpha=0.9)
legend_dot2 = Line2D([0], [0], marker='o', color='w', label="Moran's I",
                     markerfacecolor=sns.color_palette("tab10")[1], markersize=5, alpha=0.7)

# Add the legend to the plot with dot markers
plt.legend(handles=[legend_dot1, legend_dot2], loc='upper right', fontsize=5, frameon=False)  # frameon=False removes the box around the legend

# Adjust layout and show plot
fig.tight_layout()
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4','Fig4j_H4_2_epithelial_de.svg'),dpi=300)
plt.show()

/home/thechenlab/miniconda3/envs/slidelock/lib/python3.11/site-packages/seaborn/categorical.py:510: UserWarning: *c* argument looks like a single numeric RGB or RGBA sequence, which should be avoided as value-mapping will have precedence in case its length matches with *x* & *y*.  Please use the *color* keyword-argument or provide a 2D array with a single row if you intend to specify the same RGB or RGBA value for all points.
  points = ax.scatter(sub_data["x"], sub_data["y"], color=color, **plot_kws)
/tmp/ipykernel_1346531/946030139.py:6: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  ax1.set_xticklabels(ax1.get_xticklabels(), rotation=50)
/home/thechenlab/miniconda3/envs/slidelock/lib/python3.11/site-packages/seaborn/categorical.py:510: UserWarning: *c* argument looks like a single numeric RGB or RGBA sequence, which should be avoided as value-mapping will have precedence in case its length matches with *x* & *y*.  Please use the *color* keyword-argument or provide a 2D array with a single row if you intend to specify the same RGB or RGBA value for all points.
  points = ax.scatter(sub_data["x"], sub_data["y"], color=color, **plot_kws)
No description has been provided for this image

Fig2_m¶

In [107]:
epi_de_gene4 = ['Bpifa2','Krt14','Perp','Car12']
sc.settings.figdir = '/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4'
sc.pl.spatial(adata_epithelial, color=epi_de_gene4, spot_size=70,cmap='Blues',size=1,frameon=False, save='Fig4k_epithelial_de_gene4.svg')
plt.gcf().set_size_inches(12, 3)  # Set the figure size as width=10, height=8 inches
plt.show()

WARNING: saving figure to file /mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/Fig4/showFig4k_epithelial_de_gene4.svg
No description has been provided for this image 
<Figure size 1200x300 with 0 Axes>

Muslce cells¶

In [30]:
adata_muscle = adata[(adata.obs.spot_class == 'singlet') & (adata.obs.first_type == 'Muscle_cells')].copy()
In [ ]:
mus_de = pd.read_csv('/mnt/thechenlab/Chenlei/spotmapping/fiducial/data/H4_2/type12_29_de/de_summary/sig_Muscle_cells.csv')
mus_de.columns = ['gene'] + list(mus_de.columns[1:])

FigS12_a¶

In [7]:
mus_de_gene100 = mus_de.gene[:100].values
sq.gr.spatial_neighbors(adata_muscle)
sq.gr.spatial_autocorr(
    adata_muscle,
    mode="moran",
    genes=mus_de_gene100,
    n_perms=100,
    n_jobs=10,
    use_raw=True
)

moranI_gene100 = [adata_muscle.uns["moranI"].loc[gene,'I'] for gene in mus_de_gene100]

  0%|          | 0/100 [00:00<?, ?/s]
In [10]:
from matplotlib.lines import Line2D
fig, ax1 = plt.subplots(figsize=(12, 2))
sns.stripplot(data=mus_de.iloc[:100,], x="gene", y="Z_score", ax=ax1, c=sns.color_palette("tab10")[0], alpha=0.9)
ax1.set_ylabel('C-SIDE Z-score', fontsize=7)
ax1.set_xticklabels(ax1.get_xticklabels(), rotation=50)
ax1.set_xlabel('Muscle Specific Gene', fontsize=7)
ax1.tick_params(axis='both', which='major', labelsize=5)
ax1.spines['top'].set_visible(False)
ax1.spines['right'].set_visible(False)

ax2 = plt.twinx()
sns.stripplot(x=mus_de.iloc[:100,].gene, y=moranI_gene100, ax=ax2, c=sns.color_palette("tab10")[1], alpha=0.7)
ax2.set_ylabel("Moran's I", fontsize=7)
ax2.tick_params(axis='both', which='major', labelsize=5)
ax2.spines['top'].set_visible(False)
ax2.spines['left'].set_visible(False)

# Create custom Line2D objects as legend keys to resemble dots
legend_dot1 = Line2D([0], [0], marker='o', color='w', label='C-SIDE Z-score',
                     markerfacecolor=sns.color_palette("tab10")[0], markersize=5, alpha=0.9)
legend_dot2 = Line2D([0], [0], marker='o', color='w', label="Moran's I",
                     markerfacecolor=sns.color_palette("tab10")[1], markersize=5, alpha=0.7)

# Add the legend to the plot with dot markers
plt.legend(handles=[legend_dot1, legend_dot2], loc='upper right', fontsize=5, frameon=False)  # frameon=False removes the box around the legend

# Adjust layout and show plot
fig.tight_layout()
# plt.savefig(os.path.join('/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4','FigS4j_H4_2_muscle_de.svg'),dpi=300)
plt.show()

/home/thechenlab/miniconda3/envs/slidelock/lib/python3.11/site-packages/seaborn/categorical.py:510: UserWarning: *c* argument looks like a single numeric RGB or RGBA sequence, which should be avoided as value-mapping will have precedence in case its length matches with *x* & *y*.  Please use the *color* keyword-argument or provide a 2D array with a single row if you intend to specify the same RGB or RGBA value for all points.
  points = ax.scatter(sub_data["x"], sub_data["y"], color=color, **plot_kws)
/tmp/ipykernel_1478560/2677593248.py:6: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  ax1.set_xticklabels(ax1.get_xticklabels(), rotation=50)
/home/thechenlab/miniconda3/envs/slidelock/lib/python3.11/site-packages/seaborn/categorical.py:510: UserWarning: *c* argument looks like a single numeric RGB or RGBA sequence, which should be avoided as value-mapping will have precedence in case its length matches with *x* & *y*.  Please use the *color* keyword-argument or provide a 2D array with a single row if you intend to specify the same RGB or RGBA value for all points.
  points = ax.scatter(sub_data["x"], sub_data["y"], color=color, **plot_kws)
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FigS12_b¶

In [32]:
## FigS4i
mus_de_gene8 = ['Tnnc2','Mylpf','Aldoa','Ttn','Myh3','Csrp3','Car3','Myl2']
sc.settings.figdir = '/mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4'
sc.pl.spatial(adata_muscle, color=mus_de_gene8, spot_size=70,cmap='Blues',size=1,frameon=False, save='FigS4i_muscle_de_gene8.png')
plt.gcf().set_size_inches(12, 3)  # Set the figure size as width=10, height=8 inches
plt.show()

WARNING: saving figure to file /mnt/thechenlab/Chenlei/spotmapping/fiducial/Figures/FigS4/showFigS4i_muscle_de_gene8.png
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<Figure size 1200x300 with 0 Axes>